*Target (protein/gene name): deoxyuridine triphosphatase *NCBI Gene # or RefSeq#: 29483 *Protein ID (NP or XP #) or Wolbachia#: LmjF06.0560 *Organism (including strain): Leishmania major Etiologic Risk Group (see link below): *Disease Information: dUTP works together with thymidylate synthase in the synthesis of dTMP. When the function of this enzyme is altered the concentration of dUMP is too high and excessive DNA repair cycles occur. It ultimately results in the death of the disease causing cells. Link to TDR Targets page (if present): TDR information Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.): TriTrypDB Essentiality of this protein: The Crystal Structure of the Leishmania major dUTP Complex of proteins: Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): Check the article where Essensiality is dicussed.
*EC#: 3.6.1.23 Link to BRENDA EC# page: BRENDA Info
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): -- link to Sigma (or other company) page for assay (see Sigma links below) -Spectrophotometric Assayability -- links to assay reagents (substrates) pages. --- List cost and quantity of substrate reagents, supplier, and catalog #
Current Inhibitors: alpha-beta-imido-dUTP, dUDP, dUMP. Expression Information (has it been expressed in bacterial cells): Expressed in E. coli Purification Method: No commentary on purification methods. Image of protein (PyMol with features delineated and shown separately): *Amino Acid Sequence:
>2CJE:A|PDBID|CHAIN|SEQUENCE
MKRARSANIPGAILHSLAELQDGLNAMIDPSWRAVRSLDNWALAITMESTELLDSYPWKWWKNLNATPDLANVRIELVDI
FHFSLSGAMQMRSTPDDEIPAASLKPLKEVMTTFLPAKECTSDPYGFVFFPLTDTQNAIASFRNIIQLANAYRFDVIIEC
IIYAAEDLGFNLVAYYIAKHTLNCIRQLSGYKDGSYVKVNNGVEDNSLLHNCIKDVSLDEVLDADKYVQAWNSIMANVYE
AFQIKESDRKDAERWFALAKENRLAIKA
*length of your protein in Amino Acids: 268 Residues Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 30353.6 Molar Extinction coefficient of your protein at 280 nm wavelength: 53650 TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it. *CDS Gene Sequence: No similar human structures *GC% Content for gene: *CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): *GC% Content for gene (codon optimized):
Do Not Need this info for Spring (but still copy these lines to your Target page for now) Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol) -- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences): **
*NCBI Gene # or RefSeq#: 29483
*Protein ID (NP or XP #) or Wolbachia#: LmjF06.0560
*Organism (including strain): Leishmania major
Etiologic Risk Group (see link below):
*Disease Information:
dUTP works together with thymidylate synthase in the synthesis of dTMP. When the function of this enzyme is altered the concentration of dUMP is too high and excessive DNA repair cycles occur. It ultimately results in the death of the disease causing cells.
Link to TDR Targets page (if present): TDR information
Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.): TriTrypDB
Essentiality of this protein: The Crystal Structure of the Leishmania major dUTP
Complex of proteins:
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): Check the article where Essensiality is dicussed.
*EC#: 3.6.1.23
Link to BRENDA EC# page: BRENDA Info
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
-- link to Sigma (or other company) page for assay (see Sigma links below)
-Spectrophotometric Assayability
-- links to assay reagents (substrates) pages.
--- List cost and quantity of substrate reagents, supplier, and catalog #
Structure (PDB or Homology model)
2CJE
Current Inhibitors: alpha-beta-imido-dUTP, dUDP, dUMP.
Expression Information (has it been expressed in bacterial cells): Expressed in E. coli
Purification Method: No commentary on purification methods.
Image of protein (PyMol with features delineated and shown separately):
*Amino Acid Sequence:
>2CJE:A|PDBID|CHAIN|SEQUENCE
MKRARSANIPGAILHSLAELQDGLNAMIDPSWRAVRSLDNWALAITMESTELLDSYPWKWWKNLNATPDLANVRIELVDI
FHFSLSGAMQMRSTPDDEIPAASLKPLKEVMTTFLPAKECTSDPYGFVFFPLTDTQNAIASFRNIIQLANAYRFDVIIEC
IIYAAEDLGFNLVAYYIAKHTLNCIRQLSGYKDGSYVKVNNGVEDNSLLHNCIKDVSLDEVLDADKYVQAWNSIMANVYE
AFQIKESDRKDAERWFALAKENRLAIKA
*length of your protein in Amino Acids: 268 Residues
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 30353.6
Molar Extinction coefficient of your protein at 280 nm wavelength: 53650
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
*CDS Gene Sequence: No similar human structures
*GC% Content for gene:
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
*GC% Content for gene (codon optimized):
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
**
Resources:
See ProtocolTargetDiscoveryVDS.docx for more
Etiologic Risk Group Categories (for pathogens): http://www.utexas.edu/research/rsc/ibc/agent_class.html#_Toc7238334
SIGMA-ALDRICH RESOURCES
Enzyme Explorer
http://www.sigmaaldrich.com/life-science/metabolomics/enzyme-explorer.html
Enzyme Classification Index (EC number)
http://www.sigmaaldrich.com/life-science/biochemicals/biochemical-products.html?TablePage=14573088
WolframAlpha http://www.wolframalpha.com/
DrugBank http://www.drugbank.ca/
Databases of genes/organisms:
http://www.niaid.nih.gov/Pages/default.aspx
http://eupathdb.org/eupathdb/
https://patricbrc.vbi.vt.edu/portal/portal/patric/Home
http://www.nmpdr.org/FIG/wiki/view.cgi/Main/EssentialGenes
http://tubic.tju.edu.cn/deg/
http://csgid.org/csgid/cake/pages/community_request_gateway
http://tdrtargets.org/
http://gsc.jcvi.org/status.shtml
Scientific Nomenclature page from Center for Disease Control (gene, protein names and abbreviations)
http://wwwnc.cdc.gov/eid/pages/scientific-nomenclature.htm
Gene Information:
NCBI GENE Page: http://www.ncbi.nlm.nih.gov/gene
BLAST Page: http://blast.ncbi.nlm.nih.gov/
Protein Information:
NCBI Protein Page: http://www.ncbi.nlm.nih.gov/protein
Protein Expression Website
Protein Expression Paper: SGC_ProteinProductionPurificationNatMethods2008.pdf
Primer Overlap PCR Articles
HooverLubkowski_PCRoverlapcloninggnf042.pdf
StemmerPCRoverlapGene1995.pdf
Is my target good for Virtual Screening programs?
Reynolds_THermodynamicsLigandBinding_MedChemLett2011.pdf